Brain metastases occur in approximately 15% of metastatic breast cancer patients and confer a dismal prognosis. Brain metastases are thought to increasing, particuarly among women with Her-2 positive tumors. Our goals are to identify genes that functionally contribute to brain metastatic progression and to identify and validate preclinical leads. A murine preclinical model of brain metastasis was developed using a derivative of the MDA-MB-231 breast carcinoma cell line (231-BR cells). Characterization of this line was performed. In vivo brain metastases formed by 231-BR cells have a high proliferative rate and an almost undetectable apoptosis rate. Metastases are infiltrated to various extents by microglia and reactive astrocytes. Staining on a cohort of 16 resected human brain metastases revealed similar conclusions. The data suggest that the model system is reflective of human disease. Also, the data suggest that the brain microenvironment may significantly contribute to the metastatic phenotype. The first lead investigated was Her-2. In collaboration with Ken Aldape, Her-2 was overexpressed in 37% of brain metastatic specimens, larger than historic estimates of amplification in primary tumors. At least three factors could explain the increased incidence of brain metastases in Her-2+ patients: their increased lifespan, the inability of trastuzumab (Herceptin) to cross the blood-brain barrier, and a brain metastasis promoting effect of Her-2 overexpression. We tested the hypothesis that Her-2 overexpression alters the natural history of breast cells to render them more brain metastatic. Her-2 transfectants of the 231-BR cells have been engineered and characterized in vitro and in vivo. In vitro, the Her-2 transfectants exhibited increased colonization; in vivo, Her-2 overexpression conferred a three fold increase in the number of large brain metastases, proportional to MRI detectable metastases in a human brain. No change was observed in the number of micrometastases. The data suggest that Her-2 does not impact the arrival or intravasation of tumor cells to the brain, but increases their colonization to clinically detectable metastases. We determined the efficacy of the dual EGFR/Her-2 tyrosine kinase inhibitor, lapatinib, on the brain metastatic colonization of a human breast carcinoma cell line.). In vitro, using siRNA knowdown of endogeneous EGFR, lapatinib equally inhibited the growth of 231BR cells that overexpressed either Her-2 or EGFR. However, when both receptors were overexpressed lapatinib increased growth inhibition approximately 30% compared to cells that overexpressed either Her-2 or EGFR. To determine the effect of lapatinib on brain metastatic colonization in vivo, mice received intracardiac injections of either the control or the Her-2 transfected 231BR cells. Five days later mice were randomized to vehicle, 30 or 100 mg/kg lapatinib by twice daily oral gavage. Histologic analysis was conducted to quantify both micrometastases and large metastases from 10 step sagittal sections in one hemisphere of the brain. For the control transfectant mean (95% CI) large metastases were 3.36 (2.73-3.98) per section in the vehicle controls, were unaffected by 30 mg/kg lapatinib but declined 54% to 1.56 (0.94-2.17) when treated with 100 mg/kg lapatinib (p=0.0001). The Her-2 transfectant produced 6.83 (5.86-7.79) large metastases per section. Treatment with 30 mg/kg resulted in a 53% decline to 3.21 (2.31-4.11) large metastases (p less than 0.0001), while treatment with 100 mg/kg resulted in a 50% decline to 3.44 (2.55-4.32) large metastases (p less than 0.0001). The data indicate that lapatinib can inhibit the brain colonization of dual EGFR-Her-2+ breast cancer cells. Ongoing experiments are determining the effect of lapatinib on the pHer-2 and pEGFR expression of brain metastases. The data suggest that this agent will show efficacy in adjuvant trials, now open. I am a member of the tissue committe of the Trans-ALTTO trial which will examine the adjuvant activity of lapatnib, including brain relapse. We plan to investigate lapatinib combinations to improve efficacy. To date, we have examined the effect of a VEGFR small molecule inhibitor, pazaponib. In vitro, lapatinib and pazaponib affect distinct molecular pathways in tumor cells. Lapatnib shuts down PI3K and Akt signaling while pazaponib reduces Mapk signaling. The mechanism of Mapk inhibition as well as in vivo experiments are ongoing. In collaboration with Dr. George Sledge of Indiana University, we have identified a 13 gene signature in formalin fixed paraffin embedded primary Her-2+ breast tumors for rapid relapse in the brain. Further validation experiments are underway. We conducted microarray analysis of surgically resected brain metastases of breast cancer, using laser capture microdissection, amplification and 30K cDNA arrays. These data were compared to a cohort of unmatched primary breast tumors, matched for histopathology, TNM and grade. A heat map comparing gene expression differences between brain metastses and unmatched primary tumors has been compiled and expression trends validated by QRT-PCR using an independent cohort. Of the genes validated, experiments are ongoing for hexokinase 2 (HK2 and pigment epithelium derived factor (PEDF). Of these, a cohort study was conducted of HK2 expression by immunohistochemistry. High expression of HK2 significantly correlated with poor patient survival after neurosurgery. Expression of a shRNA to HK2 has been achieved, with significant reductions in tumor cell HK2 expression in indepedent clones. Other hexokinases were not affected. Cellular ATP levels were reduced in the shRNA knockdown clones. Further the knockdown clones were sensitized in vitro to the apoptotic inducing effects of taxol and doxorubicin. In vivo experiments are ongoing. Analysis of the gene expression data indicated that 80% of the differentially expressed genes were down-regulated in the brain metastases. We asked whether a HDAC inhibitor could restore gene expression using a MDA-MB-231 human breast carcinoma cell subline selected for brain tropism. SAHA was selected from the multiple HDAC inhibitors since its structure appeared favorable for brain penetration. Treatment of mice with 100 mg/kg SAHA qd, beginning on day five postinjection, significantly reduced the number of large brain metastases. In contrast to the literature, we find no effect of SAHA on brain histone acetylation as determined by IHC, nor on apoptotic rates. We did uncover a novel mechanism of action, the induction of DNA double stand breaks. These data are being extended by Dr. Kevin Camphausen to look at interactions with radiation. We are preparing these data for publication and Merck is planning a clinical trial of SAHA.